Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor synthesized primarily by vascular endothelial cell. It functions as an anticoagulant, regulating tissue factor (TF) induced cascade of blood coagulation.
Under physiology condition, TF triggers extrinsic pathway of blood coagulation during hemostasis. TF binds factor VIIa, which is a serine protease, and forms a complex with it. The complex in turn catalyzes the conversion of another inactive protease factor X into the active protease factor Xa and subsequently activating the entire extrinsic pathway of blood coagulation.
However, TF may function undesirably under pathologic condition. It is believed that the extrinsic pathway of blood coagulation triggered by TF plays a major role in thrombogenesis associated with atherosclerosis, particularly under the condition when an atheromatous plaque ruptures. TF triggered blood coagulation also plays negative roles under other pathological conditions such as myocardial infarction, ischemia, cerebrovascular disease, pulmonary thromboembolism, deep vein thrombosis, DIC and sepsis.
TFPI is a natural inhibitor to TF and blocks the extrinsic pathway of blood coagulation. TFPI inhibits factor Xa (FXa) directly, and inactivates the activity of the complex formed by factor VIIa-TF (FVIIa-TF), either with TF in suspension or with TF expressed on the surface membranes of perturbed endothelial cells, thereby stopping the cascade process of extrinsic pathway.
Structurally, wild type TFPI is a protein with molecular weight of 42-48 KD. The protein has three N-glycosylation sites on 117, 167, 228 on it. During its expression in yeast, the protein may have one more potential glycosylation site on 174 or 175.
The TFPI molecular weight varies due to the difference of sugar moiety side chains on the glycosylation sites of the protein. Glycosylation sites and sugar moieties attaching on the sites vary in different species.
The precursor TFPI molecule includes a 28 amino acid residues signal peptide. After cleavage, it gives a mature protein with 276 amino acid residues, including the N terminal which is rich in acidic amino acid residues and is highly negative in charge, followed by 3 tandem Kunitz protease inhibitory domains and a carboxyl terminal (C terminal) which is rich in basic amino acid residues and is highly positive in charge. The 3 tandem Kunitz domains are called K1, K2 and K3 respectively. K2 domain binds Factor Xa (FXa) directly and suppresses its activity. K1 domain binds Factor VIIa/TF (F VIIa/TF) via TF-Factor VIIa complex, resulting in the formation of the quaternary complex TF/FVIIa/TFPI/FXa, which is devoid of enzymatic activity, and inhibiting blood coagulation. The function of K3 domain remains unknown.
It has been an avid research field to utilize TFPI's function as a natural anticoagulant to block blood coagulation and treat thrombogenesis such as atherosclerosis in patients.
For example, U.S. Pat. No. 5,888,968 discloses a pharmaceutical composition containing TFPI for patients suffering from in E. coli sepsis. TFPI may also be used to treat blood coagulation and lower cholesterol (Effect of Cholesterol Lowering on Intravascular Pools of TFPI and Its Anticoagulant Potential in Type II Hyperlipoproteinemia, Arteriosclerosis, Thrombosis, and Vascular Biology. 1995; 15:879-885.)
One of the major obstacles of clinical application of wild type TFPI as an anticoagulation drug is that natural TFPI's half life is short, seriously limiting its application clinically.
The other problem is that the yield of TFPI in lab production is low and unstable. TFPI is currently produced in prokaryotic expression system and eukaryotic expression system. E coli expression system expresses TFPI in inclusion bodies (U.S. Pat. No. 5,212,091, US patent application publication 20050037475). Complex processes such as renaturing the protein which is a process folding the protein according to its natural 3-dimensional structure from human source, have to be employed in order to obtain active TFPI protein after the product is expressed in a prokaryotic system. The final product yield of active TFPI in a prokaryotic system is low because of the after expression processes.
Eukaryotic expression systems such as CHO (Chinese hamster Ovary cell), BHK (Baby Hamster Kidney cell), mouse C127 and yeast Saccharomyces cereuisiae (Characterization of Human Tissue Factor Pathway Inhibitor Variants Expressed in Saccharomyces cereuisiae, The Journal Of Biological Chemistry, Vol. 268, No. 18, Issue of June 25, pp. 13344-13351, 1993) have the advantage of producing folded TFPI protein, and are currently used in lab production of TFPI. However, the protein obtained from this method is just a protein having 1-161 amino acid residues, not a full length TFPI. Furthermore, some of the eukaryotic expression systems are troublesome, with low quantity of active TFPI expression and high cost. On the other hand, no document shows that TFPI can be expressed in Pichia.pastoris. Some research indicates that Pichia.pastoris system can not be used to express TFPI.
Therefore, several issues have to be addressed before active TFPI can be used in clinical practice. Long half life TFPI (LTFPI) with no immunogenesis has to be developed, and current eukaryotic expression systems need to be improved to produce high quality and stable TFPI.